Identification of genotype–biochemical phenotype correlations associated with fructose 1,6-bisphosphatase deficiency

Fructose-1,6-bisphosphatase (FBPase) deficiency, caused by an FBP1 mutation, is an autosomal recessive disorder characterized by hypoglycemic lactic acidosis. Due to the rarity of FBPase deficiency, the mechanism by which the mutations cause enzyme activity loss still remains unclear. Here we identify compound heterozygous missense mutations of FBP1, c.491G>A (p.G164D) and c.581T>C (p.F194S), in an adult patient with hypoglycemic lactic acidosis. The G164D and F194S FBP1 mutants exhibit decreased FBP1 protein expression and a loss of FBPase enzyme activity. The biochemical phenotypes of all previously reported FBP1 missense mutations in addition to G164D and F194S are classified into three functional categories. Type 1 mutations are located at pivotal residues in enzyme activity motifs and have no effects on protein expression. Type 2 mutations structurally cluster around the substrate binding pocket and are associated with decreased protein expression due to protein misfolding. Type 3 mutations are likely nonpathogenic. These findings demonstrate a key role of protein misfolding in mediating the pathogenesis of FBPase deficiency, particularly for Type 2 mutations. This study provides important insights that certain patients with Type 2 mutations may respond to chaperone molecules.

The scientific content of the manuscript is great. The methodology and results were well described and elaborated. Classifying mutations into 3 categories was interesting. However some part of the text requires professional English language editing (highlighted in yellow). The conclusion is too lengthy.
Reviewer #2 (Remarks to the Author): The authors present a case of FBPase deficiency with genotype-phenotype correlation. They have further performed interactome analysis and attempted localization of FBPase protein and an attempt to classify previously reported missense variants. The experiments are comprehensive, well-reported and the conclusions appear logical. Major points: 1. The G294V variant has been grouped as variant without change in hydrophobicity, eben though they have acknowledged significant change in hydropathy in wild vs mutant amino acid in table 6. They may reconsider classifying the variant into the group with change in hydrophobicity, where most of the variant property are better correlated.

Minor points:
Conclusion needs a revision and should be focussed on the results of present study only. Statement in line 469 ' Thus, G207V and V325A were defined as Type 3 mutations" appears more logical when placed after line 467 ' Kikawa et al. suggested that this mutation does not play a pathogenic role in FBPase deficiency, which is consistent with findings of our study" Reviewer #3 (Remarks to the Author): The authors focused on genotype -biochemical phenotype characterisation of fructose-1,6-4 bisphosphatase deficiency performing seqeuncing appraches with function studies. Early diagnosis is very important in FBPase deficiency therefore, authors should cite the article https://doi.org/10.1515/tjb-2019-0473.
Besides that after this minor revision, this contributing study should be accepted.

Response to reviewers
Reviewer #1: Comments: The scientific content of the manuscript is great. The methodology and results were well described and elaborated. Classifying mutations into 3 categories was interesting. However some part of the text requires professional English language editing (highlighted in yellow). The conclusion is too lengthy.

Reply:
Thank you for your thoughtful comment. We concisely revised the conclusion part's description (line 512-534) and checked it with professional English language editing.

Reply:
Thank you for this excellent suggestion. Both glycine and valine are classified into hydrophobic amino acids. Therefore, the G294V variant was grouped as a variant without change in hydrophobicity in figure 3. As the reviewer suggested, there is a certain change in the hydropathy index between glycine (-0.4) and valine (4.2) in Table 1, even though glycine and valine are hydrophobic amino acids. This hydropathy difference may affect FBP1 protein misfolding. We described the hydropathy difference as the possible explanation for G294V showing type 2 features (line 494-498). Thank you for your important comment.

Minor points :
Conclusion needs a revision and should be focussed on the results of present study only. Statement in line 469 ' Thus, G207V and V325A were defined as Type 3 mutations" appears more logical when placed after line 467 ' Kikawa et al .suggested that this mutation does not play a pathogenic role in FBPase deficiency, which is consistent with findings of our study"

Reply:
Thank you for your thoughtful comment. We revised the description as the reviewer suggested (line 500-508). This description was moved to the discussion part to shorten the conclusion part.